Research Trainee Brigham and Women’s Hosp, Harvard Med. Sch. Worcester, Massachusetts, United States
Disclosure(s):
Stephen Varghese Samuel, MD: No financial relationships to disclose
Introduction/Rationale: CD8⁺T cells expressing NK cell receptors have regulatory function. We have shown that alloreactive CD4⁺T cells upregulate Qa-1 (human HLA-E) stress-peptide complex in mice, enabling elimination by Qa-1–restricted Ly49⁺CD8⁺Tregs and promoting graft survival. We hypothesized that analogous human CD8⁺Tregs exist, express killer immunoglobulin-like receptor (KIR), are enriched in graft rejection, can be expanded, and suppress HLA-E–upregulated alloimmune CD4⁺ T cells.
Methods: Cryopreserved PBMCs from kidney transplant recipients (KTRs) with no rejection (NR), antibody-mediated rejection (AMR), acute cell-mediated rejection (ACR), lupus nephritis, and healthy controls (HC) were phenotyped by flow cytometry. KIR⁺CD8⁺T cells from healthy apheresis leukoreduction samples were used to study in vitro expansion and CD4⁺T-cell suppression. In human kidney organoid (HKO) humanized murine (NSG-BLT) model, KIR⁺CD8⁺T cells were isolated to assess ex vivo expansion and suppression of alloreactive CD4⁺T cells.
Results: Inhibitory KIR (iKIR) are homologous to murine Ly49 in CD8+ Tregs. In KTRs, frequency of iKIR⁺ CD8⁺Tregs were significantly higher in AMR than HC. KIR2DL2/3 and KIR3DL1 subsets of iKIR+CD8+Tregs were most enriched. KIR2DL2/3+KIR3DL1+CD8+Tregs(CD8+Tregs) were enriched in alloimmunity compared to HC and demonstrated effector memory phenotype. IL-15-dependent in vitro expansion of FACS-sorted CD8⁺Tregs >50-fold in 3weeks while maintaining perforin and granzyme-B expression. In vitro expanded CD8+Tregs suppressed activated HLA-E–upregulated CD4⁺T cells significantly more in the presence of peptide. In a humanized murine transplant model (HKO-NSG-BLT), we showed that ex vivo–expanded CD8⁺Tregs eliminated alloreactive CD4⁺T cells in an antigen-dependent manner.
Conclusion: In alloimmunity, KIR⁺CD8⁺Tregs form a distinct subset of T cells enriched during rejection and suppress activated CD4⁺T cells in vivo and ex vivo, supporting their development as cell therapy against allograft rejection.
